Similarity of the carbohydrate structures of H-2 and Ia glycoproteins

The glycopeptides produced by pronase digestion of two H-2K, two H-2D, and three Ia glycoprotein antigens were examined for size and charge. The glycopeptides derived from all of the antigens examined were found to have m.w. of 3250 +/- 200 daltons with a similar and variable composition of sialic acid residues. These data, when combined with the similarity in monosaccharide incorporation, suggest that the general parameters of the carbohydrate structure of the Ia glycoproteins from different I subregions and H-2 glycoproteins are highly similar if not identical.     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

The role of Ia molecules in the activation of T lymphocytes. I. The activation of an IL 1-dependent IL 2-producing T cell hybridoma by Con A requires an interaction, which is not H-2-restricted, with an Ia-bearing accessory cell

A model of accessory cell-dependent lectin-mediated T cell activation was investigated by utilizing a mitogen-inducible T cell hybridoma. A continuous MHC-restricted antigen-specific T cell line was fused with the azaguanine-resistant AKR thymoma BW5147. A hybrid, RF1.16B, was identified that is minimally inducible by Con A stimulation alone but is stimulated by Con A in the presence of T cell-depleted accessory cells to produce interleukin 2. The accessory cell function can be replaced by the monokine interleukin 1. Thus the lectin is a sufficient trigger for the hybrid in the absence of MHC restriction elements. The accessory cell function from splenocytes is provided by a non-B, non-T, predominantly Ia-bearing radioresistant cell. The interaction between the RF1.16B hybrid and the accessory cell population is not H-2-restricted. Control experiments, including the use of a cloned source of accessory cells, ruled out contaminating T cells or direct lectin effects as an explanation for the lack of H-2 restriction. The finding that an Ia-bearing cell is required for activation in an MHC-nonrestricted manner is discussed, and a hypothesis is raised that Ia antigens may play a role in addition to that of being a restriction element.     

Role of Thy-1+ and Ia+ cells in ovarian function

Cryostat sections of anovulatory ovaries from persistent estrous rats following a single postnatal dose of testosterone and from persistent diestrous rats following long-term postnatal estradiol treatment were investigated. The indirect immunoperoxidase technique was used to localize ovarian Thy-1 and Ia glycoproteins, as well as several other cell surface markers, and the results were compared with those obtained in normal ovaries of cycling females. Folliculogenesis in persistent estrous rats proceeded up to cystic antral follicles and was associated with the occurrence of Thy-1+ stromal cells under follicular basal lamina. In contrast to normal ovaries, Thy-1+ material did not invade the basal layers of granulosa cells. There was also no association of Ia+ cells with follicular basal lamina, but Ia+ cells were usually found associated with some thecal vessels. In persistent diestrous rats folliculogenesis was significantly retarded in both advanced antrum formation and thecal development. Thy-1+ cells were usually present in theca. No Thy-1+ material was found among basal layers of granulosa cells and the depletion of thecal Ia+ cells was almost complete. We suggest that normal follicular development may be dependent on the correct effects of Thy-1+ and Ia+ cells in addition to appropriate gonadotropin and steroid stimulation. On the other hand, anovulatory syndromes following postnatal androgen or estrogen treatment might be induced by temporary direct ovarian effects disturbing the establishment of the normal relationship between follicular structures and the immune system.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

T cell Ig and mucin domain-1-mediated T cell activation requires recruitment and activation of phosphoinositide 3-kinase

Ligation of the transmembrane protein T cell Ig and mucin domain (Tim)-1 can costimulate T cell activation. Agonistic Abs to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or costimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in a lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI3K. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation.     

Influence of H-2 on the ontogenesis of a spleen cell population which lacks C3 receptors and Thy-1 antigen.

During ontogeny there are marked differences in the cellularity of the spleen of mice from different inbred strains. Data from segregation analysis demonstrate that one of the genes involved in the control of the size of the spleen at 15 days lies in the H-2 region. In contrast to previous reports, we find that the cell population under H-2 influence lacks complement receptors.     

Distinct roles of IL-1 and IL-6 in human T cell activation

We have examined the mechanisms underlying the activation of human T cells by IL-1 and IL-6. We report that PHA-stimulated accessory cell-depleted tonsillar T cells fractionated on the basis of their density show a high degree of heterogeneity in their proliferative response to these cytokines, inasmuch as small dense lymphocytes essentially fail to respond whereas large cells proliferate extensively. This differential response could be ascribed to the fact that only the large cells produced IL-2 under these circumstances, thus providing unequivocal evidence for the existence of an IL-2-mediated step in the activation of human T cells by IL-1 and IL-6. The synergy between IL-1 and IL-6 was found to result from their complementary effects on the production of and response to IL-2, with IL-1 playing a preponderant role in the induction of IL-2, and IL-6 being required, in addition to IL-1, for optimal IL-2-responsiveness. Using small tonsillar T cells, it was possible to show that, concomitant with the enhanced response to IL-2, IL-6 induced a marked increase in cell size and in protein synthesis. In the absence of other factors, this activation was not followed by entry into S phase, suggesting that the essential role of IL-6 in T cell activation is to induce the cells to move from G0 to G1, where they become more responsive to the small amounts of IL-2 induced by IL-1.     

Intracellular location of T200 and Mo1 glycoproteins in human neutrophils

Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.     

Evaluation of Ia+ tumor cell lines and peritoneal exudate macrophages as accessory cells: differential requirements for the activation of certain T cell functions

Several Ia+ (BC3A, TA3, D1B) or Ia-inducible (WEHI-3, P388D1) tumor lines were tested for accessory cell function for the activation of antigen-specific T cell proliferation and for the induction of T helper cells that help B cells in antibody production. All lines were able to induce antigen-specific T cell proliferation in an MHC-restricted way, but none activated T helper cells to soluble antigens under all conditions tested. In comparison, starch-induced peritoneal exudate macrophages induced T cell proliferation as well as T cell help. Some of the lines tested induced nonspecific suppressor cells that were Ly-2-positive and partially or completely inhibited antibody responses. The induction of suppressor cells, however, is not the reason for the failure of the tumor lines to activate T helper cells. These data indicate that antigen-specific T cell proliferation and helper activity do not necessarily correlate.     

Two roles for Ia in antigen-specific T cell activation. II. Toward a Velcro model of antigen recognition

It has been previously reported that Ia Ag on APC seems to be involved in Ag-specific T cell activation in at least two different ways: one is to associate with foreign Ag to form a neoantigenic determinant (the Ag-specific Ia function), and the second is to interact with T cells in a non-Ag-specific manner. Both Ia functions are required for T cell activation. In the present study we examined whether the T cell structures responsible for the non-Ag-specific Ia interaction were separable from the Ag-specific alpha/beta TCR. Purified protein derivative of tuberculin (PPD)-specific murine hybridoma T cells and polyclonal lymph node T cells were stimulated for IL-2 production by APC pulsed with PPD, glutaraldehyde fixed, and anti-Ia antibody treated, to provide the antigenic PPD/Ia determinant, in the presence of glutaraldehyde-fixed non-Ag-pulsed APC, to provide the non-Ag-specific Ia interactions. However, in several different approaches the T cell structures or activation signals responsible for the Ag-specific recognition and non-Ag-specific Ia interactions seemed to be associated with each other in this experimental system. First, the Ag-specific and non-Ag-specific Ia interactions with T cells were both required simultaneously to initiate T cell activation, and it was not possible to activate T cells by providing either Ia signal subsequent to the other. Second, the T cell structures responsible for the non-Ag-specific Ia interactions appeared to be clonally distributed in PPD-specific lymph node T cells. Third, another T cell hybridoma specific for bovine insulin also showed dual Ia interactions, but the specificity of the non-Ag-specific Ia function was different than that for the PPD-specific T cell response. Fourth, all subclones of PPD-specific T hybridomas that had lost Ag-specific responsiveness also lost functional non-Ag-specific Ia interactions. Taken together, these observations suggest that a single species of TCR may mediate both the Ag-specific and non-Ag-specific Ia interactions. In addition, the non-Ag-specific Ia interaction with T cells augmented the Ag-specific Ia interaction for T cell activation, indicating that both types of interactions may be involved in some T cell responses. Based on these observations, a Velcromodel depicting the synergy between the two Ia functions is proposed in which a matrix of interactions consisting of higher affinity Ag binding and lower affinity Ia-TCR associations provides cooperative sets of signals necessary for cellular activation.     

Role of accessory cell processing and presentation of shed H-2 alloantigens in allospecific cytotoxic T lymphocyte responses

The present study was undertaken to evaluate the role of accessory cell processing of MHC alloantigens in the initiation of primary allospecific CTL responses. To first determine whether antigen processing by accessory cells is involved in the initiation of allospecific CTL responses, accessory cells were retreated with the lysosomotropic drug chloroquine before their addition to culture. It was found that chloroquine pretreatment abrogated their ability to function as accessory cells only when they were of responder haplotype and had no effect when the accessory cells were of stimulator haplotype. Although accessory cells of either responder or stimulator haplotype can initiate allospecific CTL responses, we have previously demonstrated that they do so by activating distinct classes of T helper TH) cells. Indeed, the differential effects of chloroquine on accessory cells of responder or stimulator haplotypes were shown to reflect the fact that chloroquine pretreatment markedly impaired the ability of accessory cells to activate self-Ia-restricted TH cells, but had little effect on the ability of the same accessory cells to activate either allo-class I- or allo-class II-specific TH cells. We next examined the possibility that accessory cells of responder haplotype mediate alloresponses by acquiring and processing shed MHC alloantigens derived from the stimulator cell population. In these experiments, accessory cell-depleted stimulator cells were fixed with paraformaldehyde to inhibit shedding of their surface MHC alloantigens. It was observed that even though mixed stimulator cells were recognized normally by allospecific CTL precursors, they completely failed to stimulate CTL responses mediated by responder haplotype accessory cells, indicating that the function of such accessory cells is dependent upon their acquisition of shed MHC alloantigens. Taken together, the data presented in this report demonstrate that accessory cells of responder haplotype function in allospecific CTL responses by acquiring and processing shed class I MHC alloantigens, and by then presenting the processed alloantigens in association with self-Ia determinants to self-Ia-restricted TH cells. Thus, these data indicate that the self-Ia-restricted TH cells that are involved in allospecific CTL responses recognize processed class I alloantigens in association with self-Ia determinants.     

Ia antigens: markers of specific T suppressors immune to H-2 complex antigens

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunisation of the F1 hybrids of recombinant strains of mice. These antisera are shown to display the 50 per cent cytotoxic effect in vitro in the presence of complement upon lymphocyte populations immune to the H-2 complex antigens and enriched for specific suppressor T cells (SSC) by fractionation on the monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by the cross antibody absorption with T- and B-cells of mice originated from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins are found to react with both B and T cells at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSC function in the presence of complement at a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSC are established to bear both Ia-antigens, I-J and I-C on the same cell, as demonstrated by the cross antibody absorption and variation of the H-2 origin of SSC. These two markers are suggested to function differently in the SSC immune to the H-2 antigens and the I-C antigen expression on the SSC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

T cell activation via CD2 [T, gp50]: the role of accessory cells in activating resting T cells via CD2

Monoclonal antibody (MAb) GT2 defines a unique epitope on the CD2 molecule. GT2 triggers T cell mitosis in combination with any MAb directed against 9.6/T11(1) or D66, two previously defined CD2 epitopes. We have shown already that accessory cells (AC) are required for plenary T-PBL activation by any pair of Ab directed against D66 + 9.6/T11(1). In this study, we further investigated their role and found it to vary with the anti-CD2 pair used. When purified T-PBL preparation is used, the level of [3H]TdR incorporation observed with anti-(GT2 + 9.6/T11(1)) Ab was not significant; however, it did prove significant, although greatly reduced, with the other anti-CD2 pairs tested. This was due to qualitative differences in the process of T-PBL activation, and the role of AC, because: anti-(GT2 + 9.6/T11(1)) did not induce IL 2-R expression on purified T-PBL, whereas the other anti-CD2 pairs tested did; anti-(GT2 + 9.6/T11(1)) did not induce detectable IL 2 secretion from purified T-PBL, whereas the other anti-CD2 pairs tested induced a low amount; and anti-CDw18 Ab inhibited the mitogenic effect of anti-(GT2 + 9.6/T11(1)) on PBMC by preventing both IL 2-R expression and IL 2 secretion, whereas anti-CDw18 Ab enhanced the mitogenic effect of the other anti-CD2 pairs tested. Paraformaldehyde-fixed AC fully restored, and recombinant IL 1 partially restored purified T-PBL mitosis triggered by all anti-CD2 pairs tested. To induce IL 2 synthesis, the necessity to cross-link anti-CD2 Ab was demonstrated by coupling one Ab on Sepharose beads and adding the second Ab in the soluble phase: under these circumstances, anti-CD2 pairs were mitogenic solely in the presence of AC. These data can be interpreted as follows. Most anti-CD2 pairs require minimal contact between AC and T-PBL to induce plenary levels of IL 2 synthesis. When anti-(GT2 + 9.6/T11(1)) are used, additional contact is necessary, both for IL 2-R expression and IL 2 synthesis, which would include CDw18 for stabilization. We believe these differences could be related to different conformational changes on the CD2 molecule, depending on the epitope on which the antibodies bind, and could account for different signaling to T cells.     

T cell activation via CD2 [T, gp50] molecules: accessory cells are required to trigger T cell activation via CD2-D66 plus CD2-9.6/T11(1) epitopes

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.     

Thy-1: a lymphoid cell subset marker capable of delivering an activation signal to mouse T lymphocytes

Monoclonal anti-Thy-1 antibodies are capable of activating mouse T cells in the absence of an antigen-specific signal. Therefore, Thy-1 appears to be connected to an alternative signal transduction pathway, operative in thymocytes as well as in neuronal cells, since this molecule is also present on brain. Biochemical data have shown that this molecule is differentially glycosylated with respect to its cellular distribution. Structure and sequence comparisons revealed a strong homology with the immunoglobulin primordial domain. In addition, the Thy-1 glycoprotein has the particularity of being anchored to the membrane via a glycophospholipid tail. Gene transfer experiments in different cell types have been performed to analyze the mechanism of the Thy-1 pathway of activation.     

CD8-mediated type 1 antitumor responses selectively modulate endogenous differentiated and nondifferentiated T cell localization, activation, and function in progressive breast cancer

CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-gamma, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Using a murine TCR transgenic T cell/breast tumor model, we show that adoptively transferred Ag-specific Tc1 cells are more effective in delaying mammary tumor growth and progression than that of functionally distinct Tc2 cells. Donor Tc1 cells administered 7 days posttumor challenge localized and persisted at sites of primary tumor growth with antitumor responses that were dependent, in part, on effector cell-derived IFN-gamma. Tc1-mediated responses markedly enhanced the appearance and local accumulation of highly differentiated (CD44(high)) CD4 and CD8 endogenous tumor-infiltrating T cells when compared with that of untreated tumor-bearing mice. Conversely, Tc1 cell transfer markedly delayed the appearance of corresponding nondifferentiated (CD44(low)) endogenous T cells. Such cells were acutely activated as defined by coexpression of surface markers associated with TCR engagement (CD69) and early T cell activation (CD25). Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IFN-gamma-inducible protein-10 in vivo. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD4 and CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment at various stages of tumor progression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.     

Thy-1- and Ly-6-mediated lymphokine production and growth inhibition of a T cell hybridoma require co-expression of the T cell antigen receptor complex

Repetitive subcloning of an Ag-specific T cell hybridoma yielded a variant that lacked functional mRNA for the Ag receptor (Ti, T cell Ag receptor alpha/beta heterodimer) beta-chains and failed to express CD3/Ti on the cell surface. Transfection with the original Ti alpha- and beta-chain genes restored CD3/Ti expression to normal levels. Whereas the parental T cell hybridoma produced IL-2 when stimulated with mAb against CD3, Thy-1, and Ly-6, the CD3/Ti negative cell failed to do so. Reconstitution of CD3/Ti expression restored normal IL-2 production in response to these mAb. A separate response to activation, the inhibition of transformed growth, was also dependent upon co-expression of CD3/Ti. These data demonstrate that cell surface expression of CD3/Ti is required for IL-2 production and growth inhibition initiated by two distinct activating molecules, and suggest that CD3/Ti may be a final common pathway for many transmembrane activation signals.